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Recently developed tissue clearing techniques, where the tissue is embedded within a hydrogel, have revolutionized our ability to resolve fine cellular structures in nearly intact tissues. However, the slow rate of penetration of antibodies within this hydrogel-tissue matrix has become a significantly limiting factor in many experiments, as thick tissues often require weeks to months to be adequately labeled. Increasing the pore size of this matrix has been investigated as a possible solution, but with only modest success. Here, we have systematically examined the diffusional behavior of antibodies and other typically used immuno-labels within this hydrogel-tissue matrix and, surprisingly, found that infiltration occurs at rates similar to those of diffusion in free solution. Therefore, changing the pore size of the matrix would be expected to afford only limited improvement and, instead, some means of active transport is necessary. We show that an electrophoretically-driven approach decreases the delivery time of antibodies by more than 800-fold over simple diffusion, without incurring structural damage. These results, together with the high quality of the images obtained with this method, demonstrate the advantage of this approach, thus significantly broadening the practical range of samples that can now be investigated by whole-mount tissue clearing methods.

Fast immuno-labeling by electrophoretically driven infiltration for intact tissue imaging